Recently, we have found that the statins, drugs that inhibit the HMG-CoA reductase enzyme in the mevalonate pathway, stimulate osteoblast differentiation and bone formation in rodents both in vitro and in vivo. Others have found that N2-containing bisphosphonates also target this pathway, albeit at more distal steps. In this application, we plan to examine the molecular mechanisms by which the statins influence osteoblast differentiation. Recent observations from the cardiovascular field show that statins reduce risk of ischemic stroke not by cholesterol-lowering actions, but rather by their effects to enhance endothelial nitric oxide synthase (eNOS) mRNA expression and nitric oxide (NO) generation in endothelial cells, an effect mediated by isoprenoid metabolites and Rho GTPase. We propose that a similar mechanism may be responsible for statin effects on osteoblast differentiation since 1) recent published observations show that eNOS null mutant mice have decreased bone formation, 2) our preliminary studies show that inhibitors of eNOS but not iNOS block effects of statins to stimulate bone formation in bone organ cultures. Further, we propose that these effects on osteoblasts are mediated ultimately by enhancing BMP-2 transcription, which leads to osteoblast differentiation and bone formation. To determine the molecular mechanisms by which statins stimulate bone formation, we propose the following aims: (1) determine if inhibition of HMG Co-A reductase is the initial effect of the statins that is responsible for their ultimate effects on bone formation; (2) examine the hypothesis that statins stimulate bone formation by effects on eNOS expression and activity, by determining if statins increase NO production in bone cells, determining if statin effects on bones can be impaired by specific inhibitors to eNOS and iNOS, and determining if statins have effects on bone in eNOS null mutant mice; (3) determine if statin and eNOS effects on bone formation are ultimately mediated through enhanced BMP-2 transcription by the use of bone cells in vitro or transgenic mice in vivo that are unresponsive to BMP-2. These experiments should provide information on the molecular mechanisms by which statins and inhibition of HMG-CoA reductase affect BMP signaling and osteoblast differentiation, and will hopefully clarify regulatory mechanisms responsible for normal bone formation. [unreadable] [unreadable]